Fourier Transform Raman Spectroscopy of Photoactive Proteins with Near-Infrared Excitation

This is the publisher's version, also available electronically from http://www.opticsinfobase.org/as/abstract.cfm?URI=as-44-7-1103.The Fourier transform (FT) Raman spectroscopic treatment of the photoactive proteins bacteriorhodopsin and the photosynthetic reaction center is reported, with excitation at 1.06 μm. Excitation at this wavelength circumvents the limitations on resonance Raman spectroscopy of these proteins imposed by their photolability and by the fluorescence of free pigments or impurities. The spectra are dominated by nonresonant Raman scattering by the protein-bound pigments retinal (in bacteriorhodopsin) and bacteriopheophytin, bacteriochlorophyll, and carotenoids (in reaction centers). The relative intensities of retinylidene modes in the spectrum for nonresonant FT Raman spectroscopy of bacteriorhodopsin are nearly identical to those observed in the resonance Raman spectrum of bacteriorhodopsin

Time‐resolved two‐photon induced anisotropy decay: The rotational diffusion regime

This is the publisher's version, also available electronically from http://scitation.aip.org/content/aip/journal/jcp/101/12/10.1063/1.467908.Two‐photon excitation (TPE) of randomly oriented chromophores in solution generates an anisotropic distribution. In a previous paper [Chem. Phys. 179, 513 (1994)], the polarization dependence of the TPE signal probed by a secondary spectroscopic transition (fluorescence or transient absorption) was determined. In this paper, the time dependence of anisotropic two‐photon induced fluorescence or transient absorption signals due to rotational diffusion is treated in spherical tensor formalism. The two‐photon signal in general contains isotropic (orientation independent) and anisotropic (orientation dependent) contributions. The latter decay with up to five exponential components. Four time‐dependent anisotropy parameters can be defined and measured, allowing additional information, not available in conventional one‐photon fluorescence depolarization measurements, to be determined. The special case of one‐color TPE is discussed in particular. It is shown that by measurement of the linear and circular anisotropiesr 1(t) and r 2(t), more than one rotational correlation time can be determined in many cases, providing information on rotational diffusion parameters not readily determined by analogous one‐photon methods and leading in some cases to resolution of rotational motion about the principal diffusion axes of the molecule

Time‐resolved anisotropic coherent anti‐Stokes Raman scattering: A new probe of reorientational dynamics

This is the publisher's version, also available electronically from http://scitation.aip.org/content/aip/journal/jcp/99/10/10.1063/1.465690.A formalism for the time‐dependent anisotropic third‐order susceptibility induced by a linearly polarized excitation pulse has been derived to describe the time dependence of coherent anti‐Stokes Raman scattered(CARS) and Raman time‐resolved experiments. The third‐order susceptibility induced in a randomly oriented molecular system contains nine independent molecular parameters—three isotropic and six anisotropic—as compared to three independent parameters in an isotropic system. Methods of time‐resolved anisotropic anti‐Stokes Raman scattering (TRA CARS) are discussed as an approach to reorientational measurements. The dependence of the TRA CARS or TRA Raman signal on vibration and polarization component can in principle provide more rotational information than fluorescence depolarization or linear dichroismmeasurements and may allow more complete characterization of rotational dynamics. Results are reported for three types of TRA CARS experiments to demonstrate the capabilities of TRA CARS to study the rotational motion of molecules in liquids

Picosecond laser timing by rf phase shifting

This is the publisher's version, also available electronically from http://scitation.aip.org/content/aip/journal/rsi/61/3/10.1063/1.1141443Generation of picosecond and nanosecond time‐scale time delays in a pump‐probe laser system has been implemented without the use of an optical delay line, by rf‐phase shifting of the mode‐locking rf‐signal. The system consists of dual picosecond dye lasers pumped by synchronized mode‐locked, Q‐switched, cw Nd:YAG lasers. The phase shifter operates with better than 10‐ps precision and generates time delays from 0 to 26 ns

Variable wave vector second harmonic generation in phenanthrene

This is the publisher's version, also available electronically from http://scitation.aip.org/content/aip/journal/jcp/76/7/10.1063/1.443430.Second harmonic generation is observed in phenanthrene crystals. The experimental set is used allowed the simultaneous detection of two proton excitation (TPE) and second harmonic generation.(SHG). (AIP

Fiber Laser Based Two-Photon FRET Measurement of Calmodulin and mCherry-E0GFP Proteins

This is the peer reviewed version of the following article: Adany, P., Johnson, C. K. and Hui, R. (2012), Fiber laser based two-photon fret measurement of calmodulin and mcherry-E0GFP proteins. Microsc. Res. Tech., 75: 837–843. doi:10.1002/jemt.22002, which has been published in final form at http://doi.org/10.1002/jemt.22002. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.The speed and accuracy of Förster Resonance Energy Transfer (FRET) measurements can be improved by rapidly alternating excitation wavelengths between the donor and acceptor fluorophore. We demonstrate FRET efficiency measurements based on a fiber laser and photonic crystal fiber as the source for two-photon excitation (TPE). This system offers the potential for rapid wavelength switching with the benefits of axial optical sectioning and improved penetration depth provided by TPE. Correction of FRET signals for cross excitation and cross emission was achieved by switching the excitation wavelength with an electrically controlled modulator. Measurement speed was primarily limited by integration times required to measure fluorescence. Using this system, we measured the FRET efficiency of calmodulin labeled with Alexa Fluor 488 and Texas Red dyes. In addition, we measured two-photon induced FRET in an E0GFP-mCherry protein construct. Results from one-photon and two-photon excitation are compared to validate the rapid wavelength switched two-photon measurements

Holonomy on D-Branes

This paper shows how to construct anomaly free world sheet actions in string theory with $D$-branes. Our method is to use Deligne cohomology and bundle gerbe theory to define geometric objects which are naturally associated to $D$-branes and connections on them. The holonomy of these connections can be used to cancel global anomalies in the world sheet action.Comment: Corrections made and some typographical errors remove

Picosecond Time-Resolved Fourier Transform Raman Spectroscopy of 9,10-Diphenylanthracene in the Excited Singlet State

This is the publisher's version, also available electronically from http://www.opticsinfobase.org/as/abstract.cfm?URI=as-49-5-645.Time-resolved Fourier transform Raman spectroscopy of the highly fluorescent chromophore 9,10-diphenylanthracene (DPA) in cyclohexane and ethanol is described. Raman spectra of the first excited singlet state of DPA were obtained with 100-ps resolution at several time delays between pump pulses at 355 nm and probe pulses at 1064 nm. The near-infrared excited-state Raman scattering is enhanced by resonance with an excited-state transition of DPA. The excited-state Raman bands decay in about 5-6 ns. Evidence for interaction of the solvent with the DPA excited state is observed in the cyclohexane C-H stretching bands

Conformational Heterogeneity of a Leucine Enkephalin Analog in Aqueous Solution and SDS Micelles: Comparison of Time- Resolved FRET and Molecular Dynamics Simulations

We have undertaken time-resolved Förster resonance energy transfer (FRET) and molecular dynamics simulations to analyze conformations and conformational heterogeneity of an analog of leucine enkephalin in solution and in the presence of SDS micelles. Enkephalins are opioid pentapeptides that interact with opioid receptors in the central nervous system. We used timecorrelated single-photon counting to detect energy transfer between the N-terminal tyrosine and a tryptophan residue substituted for phenylalanine at the 4 position. FRET from Tyr to Trp was measured over a temperature range from 5°C to 55°C in aqueous solution. By taking into account Tyr rotamer interconversion rates measured previously, we determined average distances between Tyr and Trp for the two populated rotameric conformations of Tyr. Molecular dynamics simulations (100 ns) support this analysis and indicate extensive conformational heterogeneity. The simulations also predict that the FRET orientational factor is correlated with the Tyr-Trp separation. Failure to account for the correlation between orientation and distance results in errors that appear to be largely offset in YGGWL by a weighting bias inherent in the R−6 dependence of the energy-transfer rate. The Tyr lifetimes decrease upon titration of the peptides with SDS, indicating formation of compact conformations of the peptide in the micelle environment. This result is consistent with the conjecture that the lipid environment may induce formation of bioactive conformations of the peptide

FRET-FCS Detection of Intra-Lobe Dynamics in Calmodulin

Fluorescence correlation spectroscopy (FCS) can be coupled with Förster resonance energy transfer (FRET) to detect intramolecular dynamics of proteins on the microsecond time scale. Here we describe application of FRET-FCS to detect fluctuations within the N-terminal and C-terminal domains of the Ca2+-signaling protein calmodulin. Intramolecular fluctuations were resolved byglobal fitting of the two fluorescence autocorrelation functions (green-green and red-red) together with the two cross-correlation functions (green-red and red-green). To match the Förster radius forFRET to the dimensions of the N-terminal and C-terminal domains, a near-infrared acceptor fluorophore (Atto 740) was coupled with a green-emitting donor (Alexa Fluor 488). Fluctuations were detected in both domains on the time scale of 30 to 40 μs. In the N-terminal domain, the amplitude of the fluctuations was dependent on occupancy of Ca2+ binding sites. A high amplitude of dynamics in apo-calmodulin (in the absence of Ca2+) was nearly abolished at a high Ca2+ concentration. For the C-terminal domain the dynamic amplitude changed little with Ca2+ concentration. The Ca2+ dependence of dynamics for the N-terminal domain suggests that the fluctuations detected by FCS in the N-terminal domain are coupled to the opening and closing of the EF-hand Ca2+-binding loops